Morphological Differentiation Of Cells Research Articles

Modulating Golgi Stress Signaling Ameliorates Cell Morphological Phenotypes Induced by CHMP2B with Frontotemporal Dementia-Associated p.Asp148Tyr.

Some charged multivesicular body protein 2B (CHMP2B) mutations are associated with autosomal-dominant neurodegenerative frontotemporal dementia and/or amyotrophic lateral sclerosis type 7 (FTDALS7). The main aim of this study is to clarify the relationship between the expression of mutated CHMP2B protein displaying FTD symptoms and defective neuronal differentiation. First, we illustrate that the expression of CHMP2B with the Asp148Tyr (D148Y) mutation, which preferentially displays FTD phenotypes, blunts neurite process elongation in rat primary cortical neurons. Similar results were observed in the N1E-115 cell line, a model that undergoes neurite elongation. Second, these effects were also accompanied by changes in neuronal differentiation marker protein expression. Third, wild-type CHMP2B protein was indeed localized in the endosomal sorting complexes required to transport (ESCRT)-like structures throughout the cytoplasm. In contrast, CHMP2B with the D148Y mutation exhibited aggregation-like structures and accumulated in the Golgi body. Fourth, among currently known Golgi stress regulators, the expression levels of Hsp47, which has protective effects on the Golgi body, were decreased in cells expressing CHMP2B with the D148Y mutation. Fifth, Arf4, another Golgi stress-signaling molecule, was increased in mutant-expressing cells. Finally, when transfecting Hsp47 or knocking down Arf4 with small interfering (si)RNA, cellular phenotypes in mutant-expressing cells were recovered. These results suggest that CHMP2B with the D148Y mutation, acting through Golgi stress signaling, is negatively involved in the regulation of neuronal cell morphological differentiation, providing evidence that a molecule controlling Golgi stress may be one of the potential FTD therapeutic targets at the molecular and cellular levels.

CRISPR/CasRx-Mediated Knockdown of Rab7B Restores Incomplete Cell Shape Induced by Pelizaeus-Merzbacher Disease-Associated PLP1 p.Ala243Val.

Pelizaeus-Merzbacher disease (PMD, currently known as hypomyelinating leukodystrophy type 1 [HLD1]) is a hereditary hypomyelinating and/or demyelinating disease associated with the proteolipid protein 1 (plp1) gene in the central nervous system (CNS). One of the major causes of this condition is incomplete or defective oligodendroglial cell myelin sheath formation triggered by endoplasmic reticulum (ER) stress and subsequent unfolded protein response (UPR). The HLD1-associated Ala-243-to-Val mutation (p.Ala243Val) of PLP1 is widely recognized to trigger defective oligodendroglial cell morphological differentiation, primarily due to ER stress. We have previously reported that knockdown of Rab7B (also known as Rab42), a small GTP/GDP-binding protein involved in intracellular vesicle trafficking around the lysosome, can recover chemical ER stress-induced incomplete cell shapes in the FBD-102b cell line, a model of oligodendroglial cell morphological differentiation. Here, we present findings indicating that incomplete cell shapes induced by PLP1 p.Ala243Val can be restored by knockdown of Rab7B using the clustered regularly interspaced short palindromic repeats (CRISPR) and CasRx (also known as Cas13d) system. Also, the knockdown promoted the trafficking of PLP1 p.Ala243Val to lysosome-associated membrane protein 1 (LAMP1)-positive organelles. These results highlight the unique role of Rab7B knockdown in modulating oligodendroglial cell morphological changes and potentially facilitating the transport of mutated PLP1 to LAMP1-positive organelles, suggesting its potential as a therapeutic target for alleviating HLD1 phenotypes, at least in part, at the molecular and cellular levels.

Knockdown of Rab7B, But Not of Rab7A, Which Antagonistically Regulates Oligodendroglial Cell Morphological Differentiation, Recovers Tunicamycin-Induced Defective Differentiation in FBD-102b Cells.

In the central nervous system (CNS), insulative myelin sheaths are generated from the differentiated plasma membranes of oligodendrocytes (oligodendroglial cells) and surround neuronal axons to achieve saltatory conduction. Despite the functional involvement of myelin sheaths in the CNS, the molecular mechanism by which oligodendroglial cells themselves undergo differentiation of plasma membranes remains unclear. It also remains to be explored whether their signaling mechanisms can be applied to treating diseases of the oligodendroglial cells. Here, we describe that Rab7B of Rab7 subfamily small GTPases negatively regulates oligodendroglial cell morphological differentiation using FBD-102b cells, which are model cells undergoing differentiation of oligodendroglial precursors. Knockdown of Rab7B or Rab7A by the respective specific siRNAs in cells positively or negatively regulated morphological differentiation, respectively. Consistently, these changes were supported by changes on differentiation- and myelination-related structural protein and protein kinase markers. We also found that knockdown of Rab7B has the ability to recover inhibition of morphological differentiation following tunicamycin-induced endoplasmic reticulum (ER) stress, which mimics one of the major molecular pathological causes of hereditary hypomyelinating disorders in oligodendroglial cells, such as Pelizaeus-Merzbacher disease (PMD). These results suggest that the respective molecules among very close Rab7 homologues exhibit differential roles in morphological differentiation and that knocking down Rab7B can recover defective differentiating phenotypes under ER stress, thereby adding Rab7B to the list of molecular therapeutic cues taking advantage of signaling mechanisms for oligodendroglial diseases like PMD.

Extracellular HSPA5 is autocrinally involved in the regulation of neuronal process elongation

The molecular mechanisms by which neuronal processes grow are extremely complicated, involving fine-tuned regulation of extracellular and intracellular signals. It remains to be elucidated which molecules are contained in the regulation. Herein, we report for the first time that heat shock protein family A member 5 (HSPA5, also called immunoglobulin heavy chain binding endoplasmic reticulum [ER] protein [BiP]) is secreted from mouse primary dorsal neuronal ganglion (DRG) cells or neuronal cell line N1E-115, a frequently used neuronal differentiation model. Supporting these results, HSPA5 protein was co-localized not only with ER antigen KDEL but also with intracellular vesicles such as Rab11-positive secretory vesicles. Unexpectedly, addition of HSPA5 inhibited elongation of neuronal processes, whereas neutralization of extracellular HSPA5 with the antibodies elongated processes, characterizing extracellular HSPA5 as a negative regulator of neuronal differentiation. Treatment of cells with neutralizing antibodies for low-density lipoprotein receptor (LDLR) did not have significant effects on process elongation, whereas LDLR-related protein 1 (LRP1) antibodies promoted differentiation, implying that LRP1 may act as a receptor candidate for HSPA5. Interestingly, extracellular HSPA5 was greatly decreased following treatment with tunicamycin, an ER stress inducer, illustrating that the ability to form neuronal processes could be preserved, even under stress. These results suggest that neuronal HSPA5 itself is secreted to contribute to inhibitory effects on neuronal cell morphological differentiation and can be included on the list of extracellular signaling molecules negatively controlling differentiation.

Metastatic niches in patients with high-grade neuroendocrine tumours: Apparent findings of matched triple-positive radiolabelled molecular probes by nuclear medicine sequence imaging

Nuclear medicine imaging of neuroendocrine neoplasm (NEN) patients could evidence cancer cell states related to glycolytic hypermetabolism, somatostatin (SST) receptors overexpression or norepinephrine (NE) transport system hyperactivity status of cancer cells profiles, according to the radiolabelled molecular probe used: fluorodeoxyglucose (FDG), octreotide (SST analogue) or the compound metaiodobenzylguanidine (NE analogue), respectively. NEN lesions with positive positron emission tomography with 18FDG (glucose analogue radiolabelled with fluoride-18) stablished a relationship with tumour aggressiveness and poorly differentiated cell morphology. Otherwise, NEN lesions evidencing somatostatin receptors (SSTR) overexpression or abnormal surface NE transporter system hyperactivity status of cancer cell profiles correlated with well differentiated cell morphology. Theoretically, the apparent correspondence between the triple-positive imaging of glycolytic hypermetabolism, SSTR overexpression and abnormal NE transporter system hyperactivity of overlapping cancer cell states in metastatic site of a NEN patient could be revealing a single tumorigenic subpopulation with a dynamic divergent differentiation potential. The complex events of metastasis progression included transdifferentiation from epithelial-to-mesenchymal to leave the primary neoplastic niche, probable as a collective migration of cancer cells, including cancer stem cells (CSCs), to circulating and reversibly transdifferentiating from mesenchymal-to-epithelial to settle in distant niches. Considering future research perspectives, we argued whether the apparent triple-positive matched correlative imaging with the radiolabelled molecular probes (glucose, SST and NE analogues) in patients’ metastatic niches could indicate potential biopsy sites to further investigation for the potential CSCs properties. We suggested that early treatment planning with lutetium-177-based peptide receptor radionuclide therapy, could provide for the blocking of poorly differentiated cell states at the point of differentiation to well differentiated cell states, and vice versa. In addition, specific individual cases should be considered for targeted-based therapy with radiolabelled-NE analogue, blocking dedifferentiation and transdifferentiation from abnormal NE transporter system hyperactivity status to SSTR overexpression cancer cell profile.

Hesperetin Ameliorates Inhibition of Neuronal and Oligodendroglial Cell Differentiation Phenotypes Induced by Knockdown of Rab2b, an Autism Spectrum Disorder-Associated Gene Product.

Autism spectrum disorder (ASD) is a central nervous system (CNS) neurodevelopmental disorder that includes autism, pervasive developmental disorder, and Asperger's syndrome. ASD is characterized by repetitive behaviors and social communication deficits. ASD is thought to be a multifactorial disorder with a range of genetic and environmental factors/candidates. Among such factors is the rab2b gene, although it remains unclear how Rab2b itself is related to the CNS neuronal and glial developmental disorganization observed in ASD patients. Rab2 subfamily members regulate intracellular vesicle transport between the endoplasmic reticulum and the Golgi body. To the best of our knowledge, we are the first to report that Rab2b positively regulates neuronal and glial cell morphological differentiation. Knockdown of Rab2b inhibited morphological changes in N1E-115 cells, which are often used as the neuronal cell differentiation model. These changes were accomplished with decreased expression levels of marker proteins in neuronal cells. Similar results were obtained for FBD-102b cells, which are used as the model of oligodendroglial cell morphological differentiation. In contrast, knockdown of Rab2a, which is another Rab2 family member not known to be associated with ASD, affected only oligodendroglial and not neuronal morphological changes. In contrast, treatment with hesperetin, a citrus flavonoid with various cellular protective effects, in cells recovered the defective morphological changes induced by Rab2b knockdown. These results suggest that knockdown of Rab2b inhibits differentiation in neuronal and glial cells and may be associated with pathological cellular phenotypes in ASD and that hesperetin can recover their phenotypes at the in vitro level at least.

Hypomyelinating Leukodystrophy 10 (HLD10)-Associated Mutations of PYCR2 Form Large Size Mitochondria, Inhibiting Oligodendroglial Cell Morphological Differentiation.

Hypomyelinating leukodystrophy 10 (HLD10) is an autosomal recessive disease related to myelin sheaths in the central nervous system (CNS). In the CNS, myelin sheaths are derived from differentiated plasma membranes of oligodendrocytes (oligodendroglial cells) and surround neuronal axons to achieve neuronal functions. Nucleotide mutations of the pyrroline-5-carboxylate reductase 2 (PYCR2) gene are associated with HLD10, likely due to PYCR2's loss-of-function. PYCR2 is a mitochondrial residential protein and catalyzes pyrroline-5-carboxylate to an amino acid proline. Here, we describe how each of the HLD10-associated missense mutations, Arg119-to-Cys [R119C] and Arg251-to-Cys [R251C], lead to forming large size mitochondria in the FBD-102b cell line, which is used as an oligodendroglial cell differentiation model. In contrast, the wild type proteins did not participate in the formation of large size mitochondria. Expression of each of the mutated R119C and R251C proteins in cells increased the fusion abilities in mitochondria and decreased their fission abilities relatively. The respective mutant proteins, but not wild type proteins also decreased the activities of mitochondria. While cells expressing the wild type proteins exhibited differentiated phenotypes with widespread membranes and increased expression levels of differentiation marker proteins following the induction of differentiation, cells harboring each of the mutant proteins did not. Taken together, these results indicate that an HLD10-associated PYCR2 mutation leads to the formation of large mitochondria with decreased activities, inhibiting oligodendroglial cell morphological differentiation. These results may reveal some of the pathological mechanisms in oligodendroglial cells underlying HLD10 at the molecular and cellular levels.

CRISPR/CasRx-Mediated RNA Knockdown Reveals That ACE2 Is Involved in the Regulation of Oligodendroglial Cell Morphological Differentiation.

Angiotensin-converting enzyme 2 (ACE2) plays a role in catalyzing angiotensin II conversion to angiotensin (1–7), which often counteracts the renin-angiotensin system. ACE2 is expressed not only in the cells of peripheral tissues such as the heart and kidney, but also in those of the central nervous system (CNS). Additionally, ACE2 acts as the receptor required for the entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), whose binding leads to endocytotic recycling and possible degradation of the ACE2 proteins themselves. One of the target cells for SARS-CoV-2 in the CNS is oligodendrocytes (oligodendroglial cells), which wrap neuronal axons with their differentiated plasma membranes called myelin membranes. Here, for the first time, we describe the role of ACE2 in FBD-102b cells, which are used as the differentiation models of oligodendroglial cells. Unexpectedly, RNA knockdown of ACE2 with CasRx-mediated gRNA or the cognate siRNA promoted oligodendroglial cell morphological differentiation with increased expression or phosphorylation levels of differentiation and/or myelin marker proteins, suggesting the negative role of ACE2 in morphological differentiation. Notably, ACE2′s intracellular region preferentially interacted with the active GTP-bound form of Ras. Thus, knockdown of ACE2 relatively increased GTP-bound Ras in an affinity-precipitation assay. Indeed, inhibition of Ras resulted in decreasing both morphological differentiation and expression or phosphorylation levels of marker proteins, confirming the positive role of Ras in differentiation. These results indicate the role of ACE2 itself as a negative regulator of oligodendroglial cell morphological differentiation, newly adding ACE2 to the list of regulators of oligodendroglial morphogenesis as well as of Ras-binding proteins. These findings might help us to understand why SARS-CoV-2 causes pathological effects in the CNS.

Emerging histopathologic markers in early-stage oral tongue cancer: A systematic review and meta-analysis.

Although there are many histopathologic prognosticators, grading of early oral tongue squamous cell carcinoma (OTSCC) is still based on morphological cell differentiation which has low prognostic value. Here we summarize the emerging histopathological markers showing powerful prognostic value, but are not included in pathology reports. Using PubMed, Scopus, Ovid Medline, and Web of Science databases, a systematic literature search was preformed to identify early OTSCC studies that investigated the prognostic significance of hematoxylin–eosin‐based histopathologic markers. Our meta‐analysis showed that tumor budding was associated with overall survival (hazard ratio [HR] 2.32; 95% CI 1.40–3.84; p < 0.01) and disease‐specific survival (DSS) (1.89; 95% CI 1.13–3.15; p = 0.02). Worst pattern of invasion was associated with disease‐free survival (DFS) (1.95; 95% CI 1.04–3.64; p = 0.04). Tumor–stroma ratio was also associated with DFS (1.75, 95% CI 1.24–2.48; p < 0.01) and DSS (1.69; 95% CI 1.19–2.42; p < 0.01). Tumor budding, worst pattern of invasion, and tumor–stroma ratio have a promising prognostic value in early OTSCC. The evaluation and reporting of these markers is cost‐effective and can be incorporated in daily practice.

Hypomyelinating Leukodystrophy 8 (HLD8)-Associated Mutation of POLR3B Leads to Defective Oligodendroglial Morphological Differentiation Whose Effect Is Reversed by Ibuprofen.

POLR3B and POLR3A are the major subunits of RNA polymerase III, which synthesizes non-coding RNAs such as tRNAs and rRNAs. Nucleotide mutations of the RNA polymerase 3 subunit b (polr3b) gene are responsible for hypomyelinating leukodystrophy 8 (HLD8), which is an autosomal recessive oligodendroglial cell disease. Despite the important association between POLR3B mutation and HLD8, it remains unclear how mutated POLR3B proteins cause oligodendroglial cell abnormalities. Herein, we show that a severe HLD8-associated nonsense mutation (Arg550-to-Ter (R550X)) primarily localizes POLR3B proteins as protein aggregates into lysosomes in the FBD-102b cell line as an oligodendroglial precursor cell model. Conversely, wild type POLR3B proteins were not localized in lysosomes. Additionally, the expression of proteins with the R550X mutation in cells decreased lysosome-related signaling through the mechanistic target of rapamycin (mTOR). Cells harboring the mutant constructs did not exhibit oligodendroglial cell differentiated phenotypes, which have widespread membranes that extend from their cell body. However, cells harboring the wild type constructs exhibited differentiated phenotypes. Ibuprofen, which is a non-steroidal anti-inflammatory drug (NSAID), improved the defects in their differentiation phenotypes and signaling through mTOR. These results indicate that the HLD8-associated POLR3B proteins with the R550X mutation are localized in lysosomes, decrease mTOR signaling, and inhibit oligodendroglial cell morphological differentiation, and ibuprofen improves these cellular pathological effects. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD8 and their amelioration.

Fabrication and evaluation of antimicrobial biomimetic nanofiber coating for improved dental implant bioseal: An in vitro study.

A weak implant-soft tissue interface may lead to bacterial ingression, breakdown of underlying tissues, and eventually implant failure. This study proposes a surface modification technique of titanium alloy (Ti), using a nano-biopolymer scaffold to enhance soft tissue attachment in dental implants. Gelatin (20% w/v) embedded with 10 ± 2nm silver nanoparticles (AgNPs) was electrospun to form a gelatin electrospun mat (GEM) scaffold, bonded to Ti alloy surface using chemical surface functionalization. Antimicrobial activity of AgNPs was tested against representative Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) at 4, 24, and 48hours and after embedding in scaffold at 48 hours. Cytotoxicity analysis (MTT assay) was performed using the 3T3 mouse fibroblast cell line at 24 and 72 hours for two groups: control (unmodified Ti disc) and experimental (GEM embedded with AgNPs); and further validated by scanning electron microscopy. The AgNPs-embedded GEM showed good antimicrobial activity at 48 hours, with the AgNPs showing complete (99.99%) inhibition of bacterial colony counts at 24 and 48 hours. Cell viability and proliferation over the GEM modified Ti discs were seen to be significantly increased (P&lt;0.05) at 72 hours as compared with control. SEM images revealed intimate spreading of fibroblasts, with differentiated cell morphology and pseudopodial processes, indicative of enhanced fibroblastic adhesion, growth, and differentiation over the scaffold. Results show good antifouling properties and biocompatibility of the fabricated coating, making it a promising strategy to reduce postoperative infections and peri-implant diseases in Ti dental implants.

Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen.

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD−102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.

Hypomyelinating Leukodystrophy 15 (HLD15)-Associated Mutation of EPRS1 Leads to Its Polymeric Aggregation in Rab7-Positive Vesicle Structures, Inhibiting Oligodendroglial Cell Morphological Differentiation.

Pelizaeus–Merzbacher disease (PMD), also known as hypomyelinating leukodystrophy 1 (HLD1), is an X-linked recessive disease affecting in the central nervous system (CNS). The gene responsible for HLD1 encodes proteolipid protein 1 (plp1), which is the major myelin structural protein produced by oligodendroglial cells (oligodendrocytes). HLD15 is an autosomal recessive disease affecting the glutamyl-prolyl-aminoacyl-tRNA synthetase 1 (eprs1) gene, whose product, the EPRS1 protein, is a bifunctional aminoacyl-tRNA synthetase that is localized throughout cell bodies and that catalyzes the aminoacylation of glutamic acid and proline tRNA species. Here, we show that the HLD15-associated nonsense mutation of Arg339-to-Ter (R339X) localizes EPRS1 proteins as polymeric aggregates into Rab7-positive vesicle structures in mouse oligodendroglial FBD-102b cells. Wild-type proteins, in contrast, are distributed throughout the cell bodies. Expression of the R339X mutant proteins, but not the wild-type proteins, in cells induces strong signals regulating Rab7. Whereas cells expressing the wild-type proteins exhibited phenotypes with myelin web-like structures bearing processes following the induction of differentiation, cells expressing the R339X mutant proteins did not. These results indicate that HLD15-associated EPRS1 mutant proteins are localized in Rab7-positive vesicle structures where they modulate Rab7 regulatory signaling, inhibiting cell morphological differentiation. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD15.

Ribociclib enhances infigratinib-induced cancer cell differentiation and delays resistance in FGFR-driven hepatocellular carcinoma.

Background & AimsInfigratinib is a pan‐FGFR (fibroblast growth factor receptor) inhibitor that has shown encouraging activity in FGFR‐dependent hepatocellular carcinoma (HCC) models. However, long‐term treatment results in the emergence of resistant colonies. We sought to understand the mechanisms behind infigratinib‐induced tumour cell differentiation and resistance and to explore the potential of adding the CDK4/6 inhibitor ribociclib to prolong cell differentiation.MethodsNine high and three low FGFR1‐3‐expressing HCC patient‐derived xenograft (PDX) tumours were subcutaneously implanted into SCID mice and subsequently treated with either infigratinib alone or in combination with ribociclib. Tumour tissues were then subjected to immunohistochemistry to assess cell differentiation, as indicated by the cytoplasmic‐to‐nuclear ratio and markers such as CYP3A4, HNF4α and albumin. Western blot analyses were performed to investigate the signalling pathways involved.ResultsInfigratinib induced cell differentiation in FGFR1‐3‐dependent HCC PDX models, as indicated by an increase in the cytoplasmic/nuclear ratio and an increase in CYP3A4, HNF4α and albumin. Resistant colonies emerged in long‐term treatment, characterised by a reversal of differentiated cell morphology, a reduction in the cytoplasmic‐to‐nuclear ratio and a loss of differentiation markers. Western blot analyses identified an increase in the CDK4/Cdc2/Rb pathway. The addition of ribociclib effectively blocked this pathway and reversed resistance to infigratinib, resulting in prolonged cell differentiation and growth inhibition.ConclusionsOur findings demonstrate that the combined inhibition of FGFR/CDK4/6 pathways is highly effective in providing long‐lasting tumour growth inhibition and cell differentiation and reducing drug resistance. Therefore, further clinical investigations in patients with FGFR1‐3‐dependant HCC are warranted.

PP1C and PP2A are p70S6K Phosphatases Whose Inhibition Ameliorates HLD12-Associated Inhibition of Oligodendroglial Cell Morphological Differentiation.

Myelin sheaths created by oligodendroglial cells encase neuronal axons to achieve saltatory conduction and protect axons. Pelizaeus-Merzbacher disease (PMD) is a prototypic, hereditary demyelinating oligodendroglial disease of the central nervous system (CNS), and is currently known as hypomyelinating leukodystrophy 1 (HLD1). HLD12 is an autosomal recessive disorder responsible for the gene that encodes vacuolar protein sorting-associated protein 11 homolog (VPS11). VPS11 is a member of the molecular group controlling the early endosome antigen 1 (EEA1)- and Rab7-positive vesicle-mediated protein trafficking to the lysosomal compartments. Herein, we show that the HLD12-associated Cys846-to-Gly (C846G) mutation of VPS11 leads to its aggregate formation with downregulated signaling through 70 kDa S6 protein kinase (p70S6K) in the oligodendroglial cell line FBD-102b as the model. In contrast, wild-type proteins are localized in both EEA1- and Rab7-positive vesicles. Cells harboring the C846G mutant constructs decrease differentiated phenotypes with web-like structures following differentiation, whereas parental cells exhibit them suitably. It is of note that we identify PP1C and PP2A as the protein phosphatases for phosphorylated Thr-389 of p70S6K essential for kinase activation in cells. The respective knockdown experiments or inhibitor treatment stimulates phosphorylation of p70S6K and ameliorates the inhibition of morphological differentiation, as well as the formation of protein aggregates. These results indicate that inhibition of p70S6K phosphatases PP1C and PP2A improves the defective morphological differentiation associated with HLD12 mutation, thereby hinting at amelioration based on a possible molecular and cellular pathological mechanism underlying HLD12.

PP1C and PP2A are p70S6K phosphatases whose inhibition ameliorates HLD12-associated inhibition of oligodendroglial cell morphological differentiation

PP1C and PP2A are p70S6K phosphatases whose inhibition ameliorates HLD12-associated inhibition of oligodendroglial cell morphological differentiation

Differentiation of endometrial stem cells into insulin-producing cells using signaling molecules and zinc oxide nanoparticles, and three-dimensional culture on nanofibrous scaffolds.

Diabetes mellitus is the most common metabolic disorder with a high mortality and morbidity rate. A new promising strategy to treat DM is pancreatic tissue engineering. We described a 3D culture system accompanied by signaling factors to differentiate hEnSCs into IPCs in the presence of nZnO. We isolated EnSCs and cultured it in DMEM/F12 medium. Nanofibrous PLA/Cs scaffold was prepared through the electrospinning method. The morphological properties of the scaffolds and cells were evaluated by SEM. MTT assay was used to investigate the metabolic activity of the hEnSCs cultured on the scaffolds and a four-stage protocol was applied to differentiate hEnSCs. The differentiated cells were tested for pancreatic markers by immunocytochemistry, qRT-PCR and DTZ staining. The results of this study revealed that hEnSCs cultured on PLA/Cs scaffold and treated with nZnO can efficiently differentiate into IPCs. The examination of differentiated cell morphology showed their near similarity with pancreatic islet cells, and DTZ staining emphasized the presence of insulin granules inside their cytoplasm. Moreover, qRT-PCR and immunofluorescent staining results showed the efficient expression of specific gene markers of IPCs in resultant differentiated cells. Moreover, PLA/CS and nZnO were able to provide a good nanoenvironment for the differentiation of hEnSCs into IPCS the in presence of other molecules.

Structural tuning of acridones for developing anticancer agents targeting dihydrofolate reductase.

Targeting dihydrofolate reductase, here, we report the tumor growth inhibitory activity of substituted acridones. The screening of the molecules over 60 cell line panel of human cancer cells identified (S)-oxiran-2-ylmethyl 9-oxo-9,10-dihydroacridine-4-carboxylate (19) with average GI50 0.3 μM. The specificity of the compound to CCRF-CEM, MOLT-4 and SR cell lines of leukemia and SW-620, SF268, LOXIMVI, ACHN and MCF7 cancerous cells exhibiting GI50 in the nM range was observed. C6 Glioma cells treated with compound 19 showed differentiated cell morphology and cell cycle arrest in G2/M phase. The interactions of the compound with dihydrofolate reductase were ascertained with the help of enzyme immunoassays, molecular docking and molecular dynamic studies.

EPIGENETIC DYSREGULATION OF CEBPE ENHANCER FUNCTION IN MLL-REARRANGED LEUKEMIA

Mixed lineage leukemia (MLL)-rearranged leukemia is characterized by the presence of MLL fusion proteins that mediate differentiation blockage through aberrant epigenetic regulation. CCAAT enhancer binding proteins (CEBPs) are key transcription factors involved in myeloid lineage commitment and maturation, among which CEBPE is crucial in terminal differentiation of granulocytes/macrophages. Here we report that CEBPE expression is significantly lower in human MLL acute myeloid leukemia (AML) cell lines (HL60, NB4, U937 and Kasumi-1), when compared to the non-MLL AML cell lines (ML-2, MV4:11, Monomac-6 and THP-1). Importantly, TCGA dataset shows that low CEBPE expression is significantly associated with poor survival of AML patients. We further identified the putative CEBPE enhancer and validated its interaction with promoter by chromatin conformation capture (3C) assay. Strikingly, DNA hypermethylation is observed specially at the CEBPE enhancer in MLL AML cells. We further demonstrated an induction of CEBPE and myeloid marker CD11b in MLL AML cells treated with DNA methylation inhibitor 5’-aza-cytidine (5’-aza-C), suggesting the crucial epigenetic regulatory role of CEBPE enhancer. While all-trans retinoid acid failed to trigger cell differentiation in MLL AML cells, co-treatment with 5’-aza-C resulted in strong induction of CEBPE with terminal differentiated cell morphology, compared to 5’-aza-C treatment per se. It therefore suggests that de-repression of CEBPE by removal of enhancer DNA methylation may prone MLL AML cells to undergo differentiation upon chemical/pharmaceutical induction. Collectively, our work showed that aberrant DNA methylation mediated by MLL fusion proteins represses CEBPE expression through altering its enhancer activity, which subsequently leads to cell differentiation blockage in MLL AML cells.

Changes in hemocytes of Biomphalaria glabrata infected with Echinostoma paraensei and exposed to glyphosate-based herbicide.

The immune system of snails is highly sensitive to pollutants, which can suppress its immune response. We investigated the effects of exposure to the glyphosate-based herbicide Roundup® Original on the snail Biomphalaria glabrata infected by the platyhelminth Echinostoma paraensei by evaluating changes in the snail's internal defense system. Four cohorts were studied: control group, infected snails, snails treated with Roundup®, and snails infected and treated with Roundup®. The hemocyte viability was assessed, morphological differentiation of cells was observed and flow cytometry was performed to determine the morphology, viability and the lectin expression profiles. The frequencies of dead hemocytes were lower in the infected group and higher in both pesticide treated groups. Three cell types were identified: blast-like cells, hyalinocytes and granulocytes. The highest number of all types of hemocytes, as well as the highest number of dead cells, were observed in the infected, pesticide-treated group. The association between infection and herbicide exposure greatly increased the frequency of dead hemocytes, suggesting that this condition impairs the internal defense system of B. glabrata making the snails more vulnerable to parasitic infections.